Compositions of bakuchiol and methods of making the same

ABSTRACT

The present invention provides compositions of bakuchiol (UP246) having low levels of impurities, particularly furanocoumarin impurities. The present invention further provides improved methods for the isolation, purification and analysis of compositions of bakuchiol. Finally, the present invention provides methods for using the purified bakuchiol compositions and formulations thereof for the prevention and treatment of various diseases and conditions mediated by cyclooxygenase (COX), lipoxygenase (LOX), minor inflammatory conditions and various microbial infections. The methods of this invention are comprised of administering to a host in need thereof an effective amount of the composition of this invention together with a pharmaceutically acceptable carrier.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/679,337, filed May 9, 2005, entitled “Generation of High PurityBakuchiol as a Therapeutic Agent,” which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions of bakuchiol and compoundsrelated thereto having low levels of impurities, particularlyfuranocoumarin impurities. The present invention provides improvedmethods for the isolation, purification and analysis of compositions ofbakuchiol. Finally, the present invention provides methods for using thepurified bakuchiol compositions and formulations thereof for theprevention and treatment of various diseases and conditions mediated bycyclooxygenase (COX), lipoxygenase (LOX), minor inflammatory conditionsand various microbial infections.

BACKGROUND OF THE INVENTION

Bakuchiol, the structure of which is illustrated below, is a phenoliccompound having a single hydroxyl group on the aromatic ring and anunsaturated hydrocarbon chain. It has been isolated from the seeds ofPsoralea. corylifolia L. (Luguminosae) and the aerial part of Psoralea.glandulosa L. (Papilionaceae).

Bakuchiol, extracted from the plant Psoralea corylifolia, has been shownto have anti-tumor, anti-oxidant (Haraguchi et al. (September 2002)Phytother Res. 16(6):539-544), cytotoxic ((December 1989) YakugakuZasshi. 109(12):962-965), anti-microbial (Newton et al. (January 2002)J. Ethnopharmacol. 79(1):57-67) and hepatoprotective activity (Cho etal. (November 2001) Planta Med. 67(8):750-751). It has also been shownto be a topoisomerase II inhibitor (Sun et al. (March 1998) J Nat. Prod.61(3):362-366). Bakuchiol inhibits PTP1B activity in a dose-dependentmanner, displaying IC₅₀ values of 20.8+/−1.9 μM (Kim et al. (January2005) Planta Med. 71(1):87-99). The preparation and in vitro evaluationof radioiodinated bakuchiol as an anti tumor agent has been reported byBatap et al. ((March 2005) Appl Radiat Isot. 62(3):389-393). Theterpenoid chain of bakuchiol has been reported to be critical to itsanti-oxidant activity (Adhikari et al. (September 2003) Chem ResToxicol. 16(9):1062-1069).

Bakuchiol has also been reported as being a useful compound for thedevelopment of antibacterial agents against oral pathogens and as havinggreat potential for use in food additives and mouthwash for preventingand treating dental caries (Katsura et al. (November 2001) AntimicrobAgents Chemother. 45(11):3009-3013). The anti-inflammatory andantipyretic activity guided fractionation of the active extracts ofPsoralea corylifolia resulted in the isolation of bakuchiol togetherwith three other active compounds: cyclobakuchiols A and B andangelicin. (Backhouse et al. (November 2001) J. Ethnopharmacol.78(1):27-31). Bakuchiol reportedly controls leukocytic functions such aseicosanoid production, migration and degranulation in the inflammatorysite and is a weak inhibitor of secretary and intracellular PLA2. Itdose-dependently reduces the formation of LTB4 and TXB2 by humanneutrophils and platelet microsomes, respectively (Ferrandiez et al.(September 1996) J Pharm Pharmacol. 48(9):975-980.). It also inhibitsthe expression of the inducible nitric oxide synthase gene via theinactivation of nuclear transcription factor-kappaB in RAW 264.7macrophages. (Pae et al. (September 2001) International Immunopharmacol.1(9-10):1849-1855). Inhibition of mitochondrial lipid peroxidation bybakuchiol has also been reported (Haraguchi et al. (August 2000) PlantaMed. 66(6):569-571). The isolation and antihyperglycemic activity ofbakuchiol isolated from Otholobium pubescens (Fabaceae) is described byKrenisky et al. in Biol Pharm Bull. (October 1999) 22(10):1137-1140).Finally, a crude extract referred to as Buguzhi agent, containingbakuchiol, as well as, a number of other coumarin type compounds hasbeen reported as promoting bone healing (US2004/0043089A1).

Bakuchiol, therefore, is a biologically active natural product having agreat deal of potential for use in the prevention and treatment ofvarious diseases and conditions. However, there are currently a numberof limitations associated with the use of this compound due primarily toits low concentration in natural sources, as well as the presence ofco-existing toxic components. One of the main problems related to theuse of bakuchiol compositions isolated from plants in the Psoralea genusis the presence of psoralens, such as psoralen and isopsoralen, thestructures of which are set forth below. Psoralens, also known asfuranocoumarins, are naturally occurring secondary metabolites inplants, including many fruits and vegetables.

A number of health risks have been associated with the handling, topicalapplication and ingestion of psoralen-containing plants and syntheticpsoralens. Psoralens are well known to be phototoxic agents, whichincrease the sensitivity of skin to ultra violet radiation and promoteskin cancer (Epstein (1999) Med. Surg. 18(4):274-284). Psoralen has beenshown to induce growth inhibition in rats (Diawara et al. (1997) CancerLett. 114(1-2):159-160). Gonadal toxicity from crude extracts ofPsoralea plants has been linked directly with the disruption of thehypothalamus-pituitary-gonadal axis (Takizawa et al. (2002) J.Toxicological Sciences 27(2):97-105). Oral administration of thepsoralens, bergapten (5-methoxypsoralen) and xanthotoxin(8-methoxypsoralen) in the diet of female rats reduced birthrates, thenumber of implantation sites, pups, corpora lutea, full and emptyuterine weight, and circulating estrogen levels in a dose-dependentmanner (Diawara et al. (1999) J. Biochem. Molecular Toxicology13(3/4):195-203). Psoralens have also been shown to induce the mRNAs ofthe liver enzymes CYP1A1 and UGT1A6, suggesting that enhanced metabolismof estrogens by psoralens may explain the reproductive toxicity and theobserved reduction of ovarian follicular function and ovulation (Diawaraet al. (May-June 2003) Pediatr Pathol Mol Med. 22(3):247-58.) Psoralenand isopsoralen account for about 0.1-2% of the dry weight of Psoraleaseeds and about 1-20% of the weight in ethanol and other organic solventcrude extracts. There remains a need for a method for removing toxiccompounds, such as psoralen and isopsoralen, as well as other coumarinsin order to enhance the purity and safety of bakuchiol compositions,particularly those isolated from plant sources.

The release and metabolism of arachidonic acid (AA) from the cellmembrane results in the generation of pro-inflammatory metabolites byseveral different pathways. Arguably, two of the most important pathwaysto inflammation are mediated by the enzymes lipoxygenase (LOX) andcyclooxygenase (COX). These are parallel pathways that result in thegeneration of leukotrienes and prostaglandins, respectively, which playimportant roles in the initiation and progression of the inflammatoryresponse. These vasoactive compounds are chemotaxins, which promoteinfiltration of inflammatory cells into tissues and serve to prolong theinflammatory response.

Inhibition of the COX enzyme is the mechanism of action attributed tomost non-steroidal anti-inflammatory drugs (NSAIDS). There are twodistinct isoforms of the COX enzyme (COX-1 and COX-2), which shareapproximately 60% sequence homology, but differ in expression profilesand function. COX-1 is a constitutive form of the enzyme, which has beenlinked to the production of physiologically important prostaglandins,which help to regulate normal physiological functions, such as plateletaggregation, protection of cell function in the stomach and maintenanceof normal kidney function. (Dannhardt and Kiefer (2001) Eur. J. Med.Chem. 36:109-26). The second isoform, COX-2, is a form of the enzymethat is inducible by pro-inflammatory cytokines, such as interleukin-1β(IL-1β) and other growth factors. (Herschmann (1994) Cancer MetastasisRev. 134:241-56; Xie et al. (1992) Drugs Dev. Res. 25:249-65). Thisisoform catalyzes the production of prostaglandin E₂ (PGE2) fromarachidonic acid (AA).

Inhibitors that demonstrate dual specificity for COX and LOX would havethe obvious benefit of inhibiting multiple pathways of arachidonic acidmetabolism. Such inhibitors would block the inflammatory effects ofprostaglandins (PG), as well as, those of multiple leukotrienes (LT) bylimiting their production. This includes the vasodilation,vasopermeability and chemotactic effects of PGE2, LTB4, LTD4 and LTE4,also known as the slow reacting substance of anaphalaxis. Of these, LTB4has the most potent chemotactic and chemokinetic effects. (Moore (1985)in Prostanoids: pharmacological, physiological and clinical relevance,Cambridge University Press, N.Y., pp. 229-230).

Because the mechanism of action of COX inhibitors overlaps that of mostconventional NSAID's, COX inhibitors are used to treat many of the samesymptoms, including pain and swelling associated with inflammation intransient skin conditions and chronic diseases in which inflammationplays a critical role. Consequently, the enzymes responsible forgenerating these mediators of inflammation have become the targets inthe development of a number of novel drugs aimed at the treatment ofinflammation, which contributes to the pathogenesis of diseases such asrheumatoid arthritis, osteoarthritis and Alzheimer's disease.

Transient skin conditions include treatment of inflammation associatedwith minor abrasions or contact dermatitis, as well as, skin conditionsthat are directly associated with the prostaglandin and leukotrienepathways, such as skin hyperpigmentation, age spots, vitilago, systemiclupus erythromatosus, psoriasis, carcinoma, melanoma, and othermammalian skin cancers. The use of COX inhibitors has been expanded toinclude diseases, such as systemic lupus erythromatosus (SLE) (Goebel etal. (1999) Chem. Res. Toxicol. 12:488-500; Patrono et al. (1985) J.Clin. Invest. 76:1011-1018), as well as rheumatic skin conditions, suchas scleroderma. COX inhibitors are also used for the relief ofinflammatory skin conditions that are not of rheumatic origin, such aspsoriasis, in which reducing the inflammation resulting from theoverproduction of prostaglandins could provide a direct benefit. (Foghet al. (1993) Acta Derm Venerologica 73:191-193). Recently overexpression of 5-lipoxygenase in the skin of patients with systemsclerosis has been reported. This has led to the suggestion that the LOXpathway may be of significance in the pathogenesis of system sclerosisand may represent a valid therapeutic target. (Kowal-Bielecka (2001)Arthritis Rheum. 44(8):1865). Finally, the increased enzymatic activityof both the COX-2 and 5-LOX at the site of allergen injections suggeststhe potential for using dual COX/LOX inhibitors to treat the symptoms ofboth the early and late phases of the skin allergic response. (Church(2002) Clin. Exp. Allergy. 32(7):1013).

Prostaglandins and leukotrienes also play important roles in thephysiological and pathological processes of wounds, burns, scald, acne,microbial infections, dermatitis, and many other diseases and conditionsof the skin. The activation of a pro-inflammatory cascade after thermalor chemical burns with significantly elevated cyclooxygenase andlipoxygenase activities are well documented and play an important rolein the development of subsequent severe symptoms and immune dysfunctionthat may lead to multiple organ failure. (Schwacha (2003) Burns 29(1):1;He (2001) J. Burn Care Rehabil. 22(1):58).

In addition to their use as anti-inflammatory agents, another potentialrole for COX inhibitors is in the treatment of cancer. Over expressionof COX has been demonstrated in various human malignancies andinhibitors of COX have been shown to be efficacious in the treatment ofanimals with skin tumors. While the mechanism of action is notcompletely understood, the over expression of COX has been shown toinhibit apoptosis and increase the invasiveness of tumorgenic celltypes. (Dempke et al. (2001) J. Can. Res. Clin. Oncol. 127:411-17; Mooreand Simmons (2000) Current Med. Chem. 7:1131-1144). Up regulated COXproduction has been implicated in the generation of actinic keratosisand squamous cell carcinoma in skin. Increased amounts of COX were alsofound in lesions produced by DNA damage. (Buckman et al. (1998)Carcinogenesis 19:723). Therefore, control of expression or proteinfunction of COX would seem to lead to a decrease in the inflammatoryresponse and the eventual progression to cancer. In fact, COX inhibitorssuch as indomethacin and Celebrex™ have been found to be effective intreating UV induced erythema and tumor formation. (Fischer (1999) Mol.Carcinog. 25:231; Pentland (1999) Carcinogenesis 20:1939). Recently, theover expression of lipoxygenase has also been shown to be related toepidermal tumor development (Muller (2002) Cancer Res. 62(16):4610) andmelanoma carcinogenesis (Winer (2002) Melanoma Res. 12(5):429). Thearachidonic acid (AA) metabolites generated from lipoxygenase pathwaysplay important roles in tumor growth related signal transductionsuggesting that that the inhibition of lipoxygenase pathways should be avalid target to prevent cancer progression. (Cuendet (2000) Drug MetabolDrug Interact 17(4):109; Steele (2003) Mutat Res. 523-524:137). Thus,the use of therapeutic agents having dual COX/LOX inhibitory activityoffers significant advantages in the chemoprevention of cancer.

Acne is a chronic disease of the pilosebaceous unit characterized byexcess production of sebum by the sebaceous glands, follicularepithelial desquamation, bacterial proliferation and inflammation.Hormone imbalance, microbial infection and inflammation are three of themajor factors associated with the onset of acne (Toombs (2005) Dermatol.Clin. 23(3):575-581; Nishijima et al. (2000) J. Dermatol.27(5):318-323). Current therapeutic agents for the prevention andtreatment of acne include anti-inflammatory agents, such as retinoids,antimicrobial agents and hormonal drugs. (Leyden (2003) J. Am. Acad.Dermatol. 49(3 Suppl):S200).

The principal bacterial species associated with acne arePropionibacterium acnes and gram-positive Staphylococcus epidermidis(Perry and Lambert (2006) Lett. Appl. Microbiol. 42(3):185-188). Currenttherapeutic agents include benzoyl peroxide and other antimicrobialdrugs, such as Ampicillin and Gentamicin (Fermandez et al. (2005) ExpertRev. Anti Infect Ther. 3(4):557-591). Unfortunately, drug resistance byboth Propionibacterium acnes and Staphylococcus epidermidis has beenreported (Nishijima et al. (2000) J. Dermatol. 27(5):318-323).

The topical application of anti-inflammatory drugs, such as retinoids(Millikan (2003) J. Am. Acad. Dermatol. 4(2):75) and the COX inhibitorsalicylic acid (Lee (2003) Dermatol Surg 29(12):1196) have also beenclinically demonstrated to be an effective and safe therapy for thetreatment of acne. Additionally, the use of nonsteroidalanti-inflammatory drugs (NSAIDs) are well documented as therapeuticagents for common and uncommon dermatoses, including acne, psoriasis,sun burn, erythema nodosum, cryoglobulinemia, Sweet's syndrome, systemicmastocytosis, urticarial, liverdoid and nodular vasculitis. (Friedman(2002) J. Cutan Med. Surg. 6(5):449).

Periodontal disease is an inflammation and infection of some or all ofthe tooth support structures (gingiva, cementum, periodontal ligament,alveolar bone and other tissues surrounding the teeth). Gingivitis(gums) and periodontitis (gums and bone) are the two main forms ofperiodontal disease. According to National Oral Information distributedby the National Institute of Dental and Craniofacial Research, anestimated 80 percent of American adults currently have some form ofperiodontal disease. Periodontal disease is initiated when a pellicleforms on a clean tooth or teeth. This pellicle attracts aerobicgram-positive bacteria (mostly actinomyces and streptococci), whichadhere to the tooth forming plaque. Within days the plaque thickens, theunderlying bacteria run out of oxygen and anaerobic motile rods andspirochetes begin to populate the subgingival area. Endotoxins releasedby the anaerobic bacteria cause inflammation, gum tissue destruction andeven bone loss. There are four primary stages of periodontal diseasethat can be characterized as indicated below. The destructive impact ofperiodontal disease goes beyond dental hygiene and health, in thatmicroscopic lesions resulting from periodontal disease have been foundin the liver, kidneys, and brain of some affected persons.

Four Stages of Periodontal Disease Grade 1 Inflammation Grade 2Inflammation, edema, gingival bleeding upon probing Grade 3Inflammation, edema, gingival bleeding upon probing, pustular discharge-- slight to moderate bone loss Grade 4 Inflammation, edema, gingivalbleeding upon probing, pustular discharge, mobility -- severe bone loss

Current methods for treating periodontal disease are limited withcontrol of the infection being the primary goal. (Genco et al. (1990) inContemporary Periodontics, The C.V. Mosby Company, St. Louis, pp.361-370.). Common anti-microbial or anti-plaque agents includechlorhexidine, Triclosan, stannous fluoride, Listerine, hydrogenperoxide, cetylpyridimiun chloride and sanguinarine alkaloids.Prescription anti-microbial mouth rinse, antiseptic chip, antibioticgel/micro-spheres, and enzyme suppressant-doxycycline are the preferrednon-mechanical/physical options to treat and control periodontaldisease. Unfortunately, there is currently no single periodontalmedication which functions to both control the inflammation as well asinhibit the infection.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

SUMMARY OF THE INVENTION

The present invention includes a novel composition of matter comprisedof bakuchiol, which is substantially free of impurities, particularlyfuranocoumarin impurities. This composition of matter is also referredto herein as UP256. In some embodiments, the composition is obtainedfrom the family of plants including, but not limited to Luguminosae,Papilionaceae, Lauraceae and Magnoliaceae. In other embodiments, thecomposition is obtained from a plant or plants selected from the genusof plants including, but not limited to Psorlea, Sassafras, Magnolia andAstractylodes. In preferred embodiments, the plant is selected from thegroup including, but not limited to Psoralea corylifolia L.(Luguminosae) or Psoralea glandulosa L. (Papilionaceae). The compositionof the invention may be obtained from the whole plant or from one ormore individual parts of the plant including, but not limited to theseeds, stems, bark, twigs, tubers, roots, root bark, young shoots,rhixomes, flowers and other reproductive organs, leaves and other aerialparts.

The amount of bakuchiol in the composition can be in the range of about14 to 100 weight percent (%) depending on the method of extraction andthe extent of purification of the crude extract. In one embodiment theamount of bakuchiol in the composition is in the range of 30% to 100%.In other embodiments the amount of bakuchiol in the composition isselected from the group consisting of at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least 90%. In a preferred embodiment, the amount ofbakuchiol in the composition is approximately 30%.

An impurity includes any substance that is unwanted in the bakuchiolcomposition. Typically, the impurities present in the bakuchiolcompositions are a result of the process employed to produce them,including both isolation from natural sources and synthetic methods. Forexample, in the isolation of bakuchiol from natural sources impuritiesinclude furanocoumarins, such as psoralen, isopsoralen and othercoumarin type components.

The present invention also includes improved methods for isolating andpurifying crude compositions of bakuchiol and related compounds obtainedfrom natural sources. The improved method for isolating and purifyingthese compositions includes the steps of extraction of the compoundsfrom a plant source, hydrolysis of the crude extract with a basicsolution, and purification by a method including but not limited tocolumn chromatography, extraction followed by crystallization, solventpartition, recrystallization and combinations thereof. Crude extractspurified in this manner are essentially free of furanocoumarinimpurities such as psoralen and isopsoralen. Thus, the potentialphototoxicity, topical irritation, carcenogenecity, and reproductivetoxicity associated with these compounds are essentially eliminated. Thepurity of these compositions following isolation and purification by themethods of the instant invention is in a range selected from about 27%to 100%.

Also included in the present invention is a method for analyzingcompositions of bakuchiol, which enables detection and quantification ofimpurities. In this embodiment of the invention, the method foranalyzing compositions of bakuchiol is comprised of the step ofanalyzing said compositions by high-pressure liquid chromatography(HPLC). Analysis by HPLC enables quantification of the variouscomponents in the mixture and also provides a means to track bakuchiol,psoralen, isopsoralen and other natural components in Psoralea plants toguide the extraction, hydrolysis and purification processes.

The present invention also includes methods for the prevention andtreatment of COX and LOX mediated diseases and conditions of the skin,mouth, teeth and gums. The method for preventing and treating COX andLOX mediated diseases and conditions of the skin, mouth, teeth and gumsis comprised of administering, preferably topically, to a host in needthereof an effective amount of a composition comprising bakuchiol, whichis substantially free of furanocumarin impurities together with apharmaceutically acceptable carrier. As noted above, the amount ofbakuchiol in the composition is in the range of 27% to 100%. Inpreferred embodiments the amount of bakuchiol in the composition isapproximately 30%. Also as noted above, in preferred embodiments, thebakuchiol is isolated from a plant or plants in the Psorlea genus ofplants.

COX and LOX mediated diseases or conditions of the skin include, but arenot limited to, acne, dandruff, sun burn, thermal burns, topical wounds,minor inflammatory conditions caused by fungal, microbial and viralinfections, vitilago, systemic lupus erythromatosus, psoriasis,carcinoma, melanoma, as well as other mammal skin cancers, skin damageresulting from exposure to ultraviolet (UV) radiation, chemicals, heat,wind and dry environments, wrinkles, saggy skin, lines and dark circlesaround the eyes, dermatitis and other allergy related conditions of theskin. COX/LOX mediated diseases and conditions of the mouth, teeth andgums, include, but not limited to periodontal diseases, oralpre-cancerous conditions, oral cancers, and other oral malignancies,sensitive gums and teeth, sequelae, pulpitis, irritation, pain andinflammation caused by the physical implantation of oral dentures,trauma, injuries, bruxism and other minor wounds in mouth, on the gumsor on the tongue, dental plague and calculus, tooth decalcification,proteolysis and caries (decay).

The present invention further includes methods for the prevention andtreatment of other COX and LOX mediated diseases and conditions,including but not limited to general joint pain and stiffness, lack ofmobility and loss of physical function due to pathological conditions ofosteoarthritis and rheumatoid arthritis, menstrual cramps,arteriosclerosis, obesity, diabetes, Alzheimer's disease, respiratoryallergic reaction, chronic venous insufficiency, psoriasis, chronictension headache, migraine headaches, inflammatory bowl disease,prostate cancer and other solid tumors.

The method for preventing and treating said COX and LOX mediateddiseases and conditions is comprised of administering to a host in needthereof an effective amount of a composition comprising bakuchiol, whichis substantially free of furanocumarin impurities together with apharmaceutically acceptable carrier. As noted above, the amount ofbakuchiol in the composition is in the range of 27% to 100%. Inpreferred embodiments the amount of bakuchiol in the composition isapproximately 30%. Also as noted above, in preferred embodiments, thebakuchiol is isolated from a plant or plants in the Psorlea genus ofplants.

Further included in the present invention are methods for the preventionand treatment of diseases and conditions of the skin, mouth, teeth orgums mediated by microbial infections, including but not limited tobacterial, viral and fungal infections, said method comprisingadministering to a host in need thereof an effective amount of apharmaceutical composition comprised of bakuchiol, which issubstantially free of furanocoumarin impurities together with apharmaceutically acceptable carrier. Diseases and conditions of theskin, mouth, gums and teeth mediated by microbial infections include,but are not limited to dandruff, acne, athletes foot, periodontaldiseases, selected from the group consisting of caries, gingivitis,periodontitis, pulpitis, periodontal conditions caused by the physicalimplantation of oral dentures, trauma, injuries, bruxism, neoplastic andother degenerative processes; material alba, pellicles, dental plagues,calculus and stains.

In one embodiment, the bacterium is selected from Propionibacteriumacnes or Staphylococcus epidermidis.

The compositions of this invention can be administered by any methodknown to one of ordinary skill in the art. The modes of administrationinclude, but are not limited to, enteral (oral) administration,parenteral (intravenous, subcutaneous, and intramuscular) administrationand topical application. In preferred embodiments the compositions areadministered topically. The method of prevention and treatment accordingto this invention comprises administering internally or topically to apatient in need thereof a therapeutically effective amount a compositioncomprised of bakuchiol, which is substantially free of impurities,particularly furanocoumarin impurities.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the HPLC chromatogram of a representative extractfrom Psoralea plants. Two furanocoumarins psoralen and isopsoralen arepresent in an equal amount in the extract.

FIG. 2 illustrate the HPLC chromatograms of Psoralea extracts before(FIG. 2A) and after (FIG. 2B) sodium hydroxide hydrolysis reaction.

FIG. 3 depicts the HPLC chromatogram of a UP256 sample (MH-258-07-01)comprised of 31% bakuchiol.

FIG. 4 depicts the HPLC chromatogram of a UP256 sample (MH-258-07-02)comprised of 41% bakuchiol.

FIG. 5 depicts the HPLC chromatogram of a UP256 sample (MH-258-12-08)comprised of 99% bakuchiol.

FIG. 6 depicts graphically a dose response curve of the inhibition ofthe activity of the enzyme 5-lipoxygenase (5-LO) by a UP256 composition(#MH-258-12-08--) relative to positive control—NDGA (-▪-).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention includes compositions of bakuchiol (UP246) havinglow levels of impurities. Included in the present invention are improvedmethods for the isolation and purification of compositions of bakuchiol.Also included in the present invention is a method for analyzingcompositions of bakuchiol, which enables the detection andquantification of various impurities. Further included in this inventionis a method for using the purified bakuchiol compositions andformulations thereof for the prevention and treatment of variousdiseases and conditions mediated by cyclooxygenase (COX), lipoxygenase(LOX), minor inflammatory conditions and various microbial infections.

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity. As such, the terms “a” or “an”, “one or more” and “atleast one” are used interchangeably herein.

“Bakuchiol” as used herein refers to the compound having the followingformula:

wherein the central double-bond may be either cis or trans. Phenoliccompounds structurally related to bakuchiol are also included withinthis definition.

As used herein the term “impurity” includes any substance that is notwanted in the bakuchiol composition, typically resulting from theisolation of bakuchiol from natural sources. The term impurity includes,but is not limited to furanocoumarin compounds selected from the groupincluding but not limited to psoralen, isopsoralen and other coumarintype impurities. Impurities also refer to impurities resulting fromsynthetic processes to obtain these compositions.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, therapeutic refers to humans as well as other animals.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult may be the alleviation of the signs, symptoms or causes of adisease or any other alteration of a biological system that is desired.

“Placebo” refers to the substitution of the pharmaceutically ortherapeutically effective dose or amount dose sufficient to induce adesired biological that may alleviate the signs, symptoms or causes of adisease with a non-active substance.

A “host” or “patient” is a living subject, human or animal, into whichthe compositions described herein are administered. Thus, the inventiondescribed herein may be used for veterinary as well as humanapplications and the terms “patient” or “host” should not be construedin a limiting manner. In the case of veterinary applications, the dosageranges can be determined as described below, taking into account thebody weight of the animal.

Note that throughout this application various citations are provided.Each citation is specifically incorporated herein by reference in itsentirety.

The present invention includes a novel composition of matter comprisedof bakuchiol, which is substantially free of impurities, particularlyfuranocoumarin impurities. This composition of matter is also referredto herein as UP256. In some embodiments, the composition is obtainedfrom a plant or plants selected from the Psorlea genus of plants. Inpreferred embodiments the plant is selected from the group including,but not limited to Psoralea corylifolia L. (Luguminosae) or Psoraleaglandulosa L. (Papilionaceae). The composition of the invention may beobtained from the whole plant or from one or more individual parts ofthe plant including, but not limited to the seeds, stems, bark, twigs,tubers, roots, root bark, young shoots, rhixomes, flowers and otherreproductive organs, leaves and other aerial parts.

The amount of bakuchiol in the composition can be in the range of about14 to 100 weight percent (%) depending on the method of extraction andthe extent of purification of the crude extract. In one embodiment theamount of bakuchiol in the composition is in the range of 30% to 100%.In other embodiments the amount of bakuchiol in the composition isselected from the group consisting of at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least 90%. In a preferred embodiment, the amount ofbakuchiol in the composition is approximately 30%.

An impurity includes any substance that is unwanted in the bakuchiolcomposition. Typically, the impurities present in the bakuchiolcompositions are a result of the process employed to produce them,including both isolation from natural sources and synthetic methods. Forexample, in the isolation of bakuchiol from natural sources impuritiesinclude furanocoumarins, such as psoralen, isopsoralen and othercoumarin type components.

The present invention also includes improved methods for isolating,analyzing and purifying crude compositions of bakuchiol obtained fromnatural sources. The improved method for isolating and purifying thesecompositions includes the steps of extraction of the compound from aplant source, hydrolysis of the crude extract with a basic solution, andpurification by a method including but not limited to columnchromatography, extraction followed by crystallization, solventpartition, recrystallization and combinations thereof. Crude extractspurified in this manner are essentially free of furanocoumarinimpurities such as psoralen and isopsoralen.

A method for analyzing compositions of bakuchiol using high pressureliquid chromatography (HPLC) is described in Example 1 (Table 1).Analysis by HPLC enables quantification of the various components in themixture and also provides a means to track bakuchiol, psoralen,isopsoralen and other natural components in Psoralea plants to guide theextraction, hydrolysis and purification processes.

The efficiency of bakuchiol extraction from plant sources was evaluatedusing six different organic solvent systems under two sets of extractionconditions as described in Example 2. The results are set forth in Table2. With reference to Table 2, it can be seen that bakuchiol can beextracted from Psoralea plants with any number of organic solventsand/or combinations thereof. The amount of bakuchiol in the variousextracts ranged from a maximum of 29.1% to 13.7% by weight. It wasdetermined that extraction with petroleum ether provided the highestpurity bakuchiol in the crude extract with good recovery. Arepresentative HPLC chromatogram of a crude extract is illustrated inFIG. 1. With reference to this figure it can be seen that the crudeextract contained bakuchiol as well as the furanocoumarin impuritiespsoralen and isopsoralen. Example 3 describes a large scale extractionwith petroleum ether at 70° C.

The efficacy of purification of crude bakuchiol extracts by columnchromatography is demonstrated in Example 4. Eight different types ofresins were evaluated specifically for their ability to separatebakuchiol from furanocoumarin impurities. Both silica gel and CG-161resins demonstrated satisfactory separation. Column chromatographicseparation of crude plant extracts on an industrial scale, however, istypically not economically feasible in that it requires expensiveequipment and reagents and experienced personnel, not to mention theextremely low loading capacity of these samples due to the complexity ofcrude plant extracts.

Examples 5-7 describe a novel, economical method for separatingbakuchiol from furanocoumarin impurities, said method comprisingtreatment of compositions containing said impurities with a base. Asillustrated by the following scheme, using NaOH for purposes ofillustration, treatment with base opens up the lactone ring of thefuranocoumarins, thereby converting them into the corresponding salts ofcarboxylic acids, which can then be easily separated from the remainderof the mixture by a variety of methods.

The basic solution can be selected from any base which can be used toopen lactone rings, including, but not limited to sodium hydroxide,potassium hydroxide, calcium hydroxide and lithium hydroxide. Thesolution can be selected to have different concentration and pH valuesto maximize the conversion to the acid salt. The reaction mixture canalso be heated under different temperature and pressures to maximize thereaction rate, efficiency and yield.

The course of the reaction can be followed by HPLC to ensure completeconversion of the coumarins into their respective carboxylic acid salts.The HPLC chromatograms of the crude composition before and afterhydrolysis are illustrated in FIGS. 2A and B. Upon completion of thereaction (as determined by HPLC), the reaction solution can be processedusing various methods, including but are not limited to columnchromatography extraction followed by crystallization, solventpartition, recrystallization or combinations thereof. Crude extractspurified in this manner are essentially free of furanocoumarinimpurities such as psoralen and isopsoralen.

With reference to Examples 5-7, upon hydrolysis under a variety ofconditions followed by solvent partition, the furanocoumarins, psoralenand isopsoralen, are effectively removed from the bakuchiol composition.Additionally, the purity of bakuchiol is enhanced from about 10-30% to30%-50%. Organic solvents that can be used for solvent partitioninclude, but are not limited to petroleum ether, ethyl acetate, ethylether, hexane, chloroform, propanol, butanol, and methylene chloride, aswell as other water immiscible organic solvents.

In one embodiment, which is described in Example 7, the crude reactionmixture is loaded directly onto a column followed by elution with apolar solvent. According to this embodiment compositions comprised of70% to 100% of bakuchiol can be obtained. In Example 7, followinghydrolysis the crude reaction mixture was loaded directly onto a CG-161cd column followed by elution with methanol to provide highly pure(approximately 99%) bakuchiol. Other types of resins that can be usedaccording to this embodiment include, but are not limited to XAD(Amerlite), CG-71/CG-161 or other type of polystyrene resins; ionexchange resins and silica gel. The column can be eluted with solventsselected from the group including, but not limited to water,methanol/water, ethanol/water, acetone/water and acetonitrile/water aswell as other combinations of polar solvents. It is worth noting thatthe loading capacity of the column after hydrolysis was much higher thanprior to hydrolysis. Additionally, the color of these highly purefuranocoumarin free, bakuchiol compositions was light brown and theywere very stable with respect to both color and composition of theactive agent, making them particularly suitable for formulation, storageand cosmetic applications.

In alternate embodiments, the crude reaction mixture can first beextracted with an organic solvent followed by further purification bychromatography and for solvent partition and/or recrystallization. Asnoted above, depending on the method of extraction and extent of furtherpurification bakuchiol compositions comprised of between about 30% and100% are readily obtainable.

The present invention includes methods for using the purified bakuchiolcompositions and formulations thereof for the prevention and treatmentof various diseases and conditions mediated by cyclooxygenase (COX),lipoxygenase (LOX), minor inflammatory conditions and various microbialinfections. The biological properties and safety of these uniquefuranocoumarin free bakuchiol compositions, referred to herein as UP256,were evaluated as described in Examples 8-11.

In Example 8, a highly pure composition of UP256 (MH-258-12-08, 99%purity) was tested for inhibition of both the COX-1 and COX-2 enzymes.UP256 showed potent inhibitory activity for both enzymes. The IC₅₀ forCOX-1 was determined to be 2.34 μM, while IC₅₀ for COX-2 was quantifiedat 78 μM. Thus, this composition provides a more balanced modulation ofthe COX-1 and COX-2 enzymes than conventional COX inhibitors. Forexample, aspirin, a COX-1 selective inhibitor, which is more than 150times more effective against COX-1 versus COX-2, causes gastrointestinalside effects. Conversely, Vioxx®, Celebrex® and Bextra®, which areselective COX-2 inhibitors having 50-200 times more potency againstCOX-2, do not cause as much gastrointestinal damage, however, theseCOX-2 selective drugs increase cardiovascular risks. The novelcomposition of matter disclosed herein on the other hand provide thebest modulation of the eicosanoid pathway without the stomach irritationcaused by COX-1 selective NSAIDs and cardiovascular risks posed by COX-2selective inhibitors.

It is also significant that the mechanism of action for COX inhibitionby UP256 is completely different than that of NSAIDs. Aspirin, Vioxx®,Celebrex® and Bextra® irreversibly bind to the COX enzyme throughcovalent bonds to form tightly bound enzyme-inhibitor complexes. Thisinteraction completely changes the active site of the enzyme and theside pocket and destroys the enzyme. (Walker and Kurumbal et al. (2001)Biochem. 357:709-718). UP256, on the other hand, inhibits the COX enzymethrough a weaker and reversible binding. In this interactive process,the structure and function of the COX enzyme are not irreversiblyaltered which results in a much better tolerance and safety profile.

Example 9 describes a LOX inhibition assay. The inhibition of LOXresults in a decrease in the accumulation of phagocytic leukotrienes,which are directly associated with the symptoms of chronic inflammation,and also reduces potential gastrointestinal side effects. Such efficacyis demonstrated in Example 9. With reference to Example 9, the highlypure UP256 composition, MH-258-12-08 (99% purity) was tested induplicate at four concentrations against the human 5-lipoxygenase (5-LOor 5-LOX) enzyme. The COX-2 inhibitory activity was confirmed bymeasurement of dose response and IC₅₀ (the concentration required toinhibit 50% of the enzyme's activity). The dose response curve isdepicted in FIG. 6. The IC₅₀ for LOX inhibition was determined to be3.41 μM. Thus, UP256 provides the additional benefit of significantlyreducing leukotriene production. This reduction in leukotrieneproduction is by far superior to traditional non-steroidalanti-inflammatory drugs such as ibuprofen.

Example 10 describes an experiment designed to determine theanti-microbial activity UP256. With reference to Example 10, UP256 wastested in duplicate at eight concentrations for the inhibition of sixdifferent microbes. It was found that UP256 inhibited two specificmicrobes, Propionibacterium acnes and gram-positive Staphylococcusepidermidis, at a minimum effective concentration of 1 μg/mL. Both ofthese microbes are directly associated with acne, dermatitis, and otherskin infections. UP256 also showed moderate inhibition of Trichophytonmentagrophytes at a concentration 30 mg/mL. No inhibition was observedfor Epidermophyton floccosum, Microsporum canis or Pityrosporum ovale.

UP256, at concentrations of 30% and 70%, was tested for acute toxicityin mice as described in Example 10. The mice tested were given an oraldaily of 2 g/kg for 14 days. Mice showed no adverse effects in terms ofweight gain and blood chemistry. Additionally, no toxicity was observedin any of the major organs tested. In conclusion, weight, blood work andhistological data, was no different for the treatment group than for thecontrol group. No adverse effects were observed in the fourteen-daystudy. Thus, it can be concluded that UP256 has a solid safety profile.

Finally, UP256 has a partition coefficient of log P=6.13. The partitioncoefficient of a chemical compound provides a thermodynamic measure ofits hydrophilicity/lipophilicity balance and thus its potentialbioavailability. Having a partition coefficient of 6.13 means thiscompound has high cell membrane penetration and bioavailability whenformulated in a delivery system.

The present invention therefore includes methods for the prevention andtreatment of COX and LOX mediated diseases and conditions of the skin,mouth, teeth and gums. The method for preventing and treating COX andLOX mediated diseases and conditions of the skin, mouth, teeth and gumsis comprised of administering, preferably topically, to a host in needthereof an effective amount of a composition comprising bakuchiol, whichis substantially free of furanocumarin impurities together with apharmaceutically acceptable carrier. As noted above, the amount ofbakuchiol in the composition is in the range of 27% to 100%. Inpreferred embodiments the amount of bakuchiol in the composition isapproximately 30%. Also as noted above, in preferred embodiments, thebakuchiol is isolated from a plant or plants in the Psorlea genus ofplants.

COX and LOX mediated diseases or conditions of the skin include, but arenot limited to, acne, dandruff, sun burn, thermal burns, topical wounds,minor inflammatory conditions caused by fungal, microbial and viralinfections, vitilago, systemic lupus erythromatosus, psoriasis,carcinoma, melanoma, as well as other mammal skin cancers, skin damageresulting from exposure to ultraviolet (UV) radiation, chemicals, heat,wind and dry environments, wrinkles, saggy skin, lines and dark circlesaround the eyes, dermatitis and other allergy related conditions of theskin. COX/LOX mediated diseases and conditions of the mouth, teeth andgums, include, but not limited to periodontal diseases, oralpre-cancerous conditions, oral cancers, and other oral malignancies,sensitive gums and teeth, sequelae, pulpitis, irritation, pain andinflammation caused by the physical implantation of oral dentures,trauma, injuries, bruxism and other minor wounds in mouth, on the gumsor on the tongue, dental plague and calculus, tooth decalcification,proteolysis and caries (decay).

The present invention further includes methods for the prevention andtreatment of other COX and LOX mediated diseases and conditions,including but not limited to general joint pain and stiffness, lack ofmobility and loss of physical function due to pathological conditions ofosteoarthritis and rheumatoid arthritis, menstrual cramps,arteriosclerosis, obesity, diabetes, Alzheimer's disease, respiratoryallergic reaction, chronic venous insufficiency, psoriasis, chronictension headache, migraine headaches, inflammatory bowl disease,prostate cancer and other solid tumors.

The method for preventing and treating said COX and LOX mediateddiseases and conditions is comprised of administering to a host in needthereof an effective amount of a composition comprising bakuchiol, whichis substantially free of furanocumarin impurities together with apharmaceutically acceptable carrier. As noted above, the amount ofbakuchiol in the composition is in the range of 27% to 100%. Inpreferred embodiments the amount of bakuchiol in the composition isapproximately 30%. Also as noted above, in preferred embodiments, thebakuchiol is isolated from a plant or plants in the Psorlea genus ofplants.

Further included in the present invention are methods for the preventionand treatment of diseases and conditions of the skin, mouth, teeth orgums mediated by microbial infections, including but not limited tobacterial, viral and fungal infections, said method comprisingadministering to a host in need thereof an effective amount of apharmaceutical composition comprised of bakuchiol, which issubstantially free of furanocoumarin impurities together with apharmaceutically acceptable carrier. Diseases and conditions of theskin, mouth, gums and teeth mediated by microbial infections include,but are not limited to dandruff, acne, athletes foot, periodontaldiseases, selected from the group consisting of caries, gingivitis,periodontitis, pulpitis, periodontal conditions caused by the physicalimplantation of oral dentures, trauma, injuries, bruxism, neoplastic andother degenerative processes; material alba, pellicles, dental plagues,calculus and stains.

In one embodiment, the bacterium is selected from Propionibacteriumacnes or Staphylococcus epidermidis.

The compositions of this invention can be administered by any methodknown to one of ordinary skill in the art. The modes of administrationinclude, but are not limited to, enteral (oral) administration,parenteral (intravenous, subcutaneous, and intramuscular) administrationand topical application. In preferred embodiments the compositions areadministered topically. The method of prevention and treatment accordingto this invention comprises administering internally or topically to apatient in need thereof a therapeutically effective amount a compositioncomprised of bakuchiol, which is substantially free of impurities,particularly furanocoumarin impurities.

The method of prevention and treatment according to this inventioncomprises administering systemically or topically to a host in needthereof a therapeutically effective amount of UP256 (bakuchiol)synthesized and/or isolated from a single plant or multiple plants and apharmaceutically acceptable carrier. The purity of the UP256 includes,but is not limited to 30% to 100%, depending on the methodology used toobtain and purify the compound. In a preferred embodiment, doses ofUP256 an efficacious, nontoxic quantity generally selected from therange of 0.001% to 100% based on total weight of the topical formulationand/or 0.001-200 mg per kilogram based on the total body weight of thehost. Persons skilled in the art using routine clinical testing are ableto determine optimum doses for the particular ailment being treated.

The present invention includes an evaluation of different compositionsof UP256 (bakuchiol) synthesized and/or isolated from a single plant ormultiple plants and a pharmaceutically acceptable carrier using enzyme,receptor, microbial and other in vitro and in vivo models to optimizethe formulation and obtain the desired physiological activity. Thecompositions of this invention can be administered by any method knownto one of ordinary skill in the art. The modes of administrationinclude, but are not limited to, enteral (oral) administration,parenteral (intravenous, subcutaneous, and intramuscular) administrationand topical application. The method of treatment according to thisinvention comprises administering internally or topically to a patientin need thereof a therapeutically effective amount of UP256 (bakuchiol)synthesized and/or isolated from a single plant or multiple plants,wherein said bakuchiol is substantially free of furanocoumarinimpurities. In a one embodiment the composition is administeredtopically. Methods for topical administration include, but are notlimited to a toothpaste, gel, ointment, mouthwash, chewing gum,tinctures, drinks and as well as other known pharmaceuticalformulations. When formulated in a toothpaste, the content of thecomposition can be in the range of 0.1 to 2 weight percent (%) ofbakuchiol.

The compositions of the present invention can be formulated aspharmaceutical compositions, which include other components such as apharmaceutically and/or cosmetically acceptable excipient, an adjuvant,and/or a carrier. For example, compositions of the present invention canbe formulated in an excipient that the host to be treated can tolerate.An excipient is an inert substance used as a diluent or vehicle for adrug. Examples of such excipients include, but are not limited to water,buffers, saline, glycerin, hydrated silica, propylene glycol, aluminumoxide, carrageenan, cellulose gum, titanium dioxide, Ringer's solution,dextrose solution, mannitol, Hank's solution, preservatives and otheraqueous physiologically balanced salt solutions. Nonaqueous vehicles,such as fixed oils, sesame oil, ethyl oleate, or triglycerides may alsobe used. Other useful formulations include suspensions containingviscosity enhancing agents, such as sodium carboxymethylcellulose,sorbitol, or dextran. Excipients can also contain minor amounts ofadditives, such as EDTA, disodium DDTA, BHA, BHT, diammonium citrate,nordihydroguaiaretic acid, propyl gallate, sodium gluconate, Sodiummetabisulfite, t-butyl hydroquinone, SnCl₂, H₂O₂, and2,4,5-trihydroxybutyrophenone, vitamin C vitamin E and other substancesthat enhance isotonicity and chemical stability. Examples of substancesfor adjusting pH of the formulation include sodium hydroxide, sodiumcarbonate, sodium bicarbonate, pentasodium triphosphate, tetrasodiumpyrophosphate, sodium lauryl sulfate, calcium peroxide, phosphatebuffer, bicarbonate buffer, tris buffer, histidine, citrate, andglycine, or mixtures thereof, while examples of flavors include, but arenot limited to thimerosal, m- or o-cresol, formalin, fruit extracts andbenzyl alcohol. Standard formulations can either be liquid or solids,which can be taken up in a suitable liquid as a suspension or solutionfor administration. Thus, in a non-liquid formulation, the excipient cancomprise dextrose, human serum albumin, preservatives, etc., to whichsterile water or saline can be added prior to administration.

In one embodiment of the present invention, the composition can alsoinclude an adjuvant or a carrier. Adjuvants are typically substancesthat generally enhance the biological response of a mammal to a specificbioactive agent. Suitable adjuvants include, but are not limited to,Freund's adjuvant; other bacterial cell wall components; aluminum,calcium, copper, iron, zinc, magnesium, stannous based salts; silica;polynucleotides; toxoids; serum proteins; viral coat proteins; otherbacterial-derived preparations; gamma interferon; block copolymeradjuvants, such as Hunter's Titermax adjuvant (Vaxcel™, Inc. Norcross,Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives, such as Quil A(available from Superfos Biosector A/S, Denmark). Carriers are typicallycompounds that increase the half-life of a therapeutic composition inthe treated host. Suitable carriers include, but are not limited to,polymeric controlled release formulations, biodegradable implants,liposomes, bacteria, viruses, oils, esters, and glycols.

In one embodiment, the composition is prepared as a controlled releaseformulation, which slowly releases the composition of the presentinvention into the host. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehicles will beknown to those skilled in the art. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

The therapeutic agents of the instant invention are administeredtopically by any suitable means, known to those of skill in the art fortopically administering therapeutic compositions including, but notlimited to as an ointment, gel, lotion, or cream base or as an emulsion,as a patch, dressing or mask, a nonsticking gauze, a bandage, a swab ora cloth wipe. Such topical application can be locally administered toany affected area, using any standard means known for topicaladministration. A therapeutic composition can be administered in avariety of unit dosage forms depending upon the method ofadministration. For particular modes of delivery, a therapeuticcomposition of the present invention can be formulated in an excipientof the present invention. A therapeutic reagent of the present inventioncan be administered to any host, preferably to mammals, and morepreferably to humans. The particular mode of administration will dependon the condition to be treated.

In one embodiment, a suitable ointment is comprised of the desiredconcentration of UP256 (bakuchiol) that is an efficacious, nontoxicquantity generally selected from the range of 0.001% to 100% based ontotal weight of the topical formulation, from 65% to 100% (preferably75% to 96%) of white soft paraffin, from 0% to 15% of liquid paraffin,and from 0% to 7% (preferably 3 to 7%) of lanolin or a derivative ofsynthetic equivalent thereof. In another embodiment the ointment maycomprise a polyethylene-liquid paraffin matrix.

In one embodiment, a suitable cream is comprised of an emulsifyingsystem together with the desired concentration of UP256 (bakuchiol)synthesized and/or isolated from a single plant or multiple plants asprovided above. The emulsifying system is preferably comprised of from 2to 10% of polyoxyethylene alcohols (e.g. the mixture available under thetrademark CetomacrogolTM1000), from 10 to 25% of stearyl alcohol, from20 to 60% of liquid paraffin, and from 10 to 65% of water; together withone or more preservatives, for example from 0.1 to 1% ofN,N″-methylenebis[N′-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea](available under the name Imidurea USNF), from 0.1 to 1% of alkyl4-hydroxybenzoates (for example the mixture available from NipaLaboratories under the trade mark Nipastat), from 0.01 to 0.1% of sodiumbutyl 4-hydroxybenzoate (available from Nipa Laboratories under thetrade mark Nipabutyl sodium), and from 0.1 to 2% of phenoxyethanol.

In one embodiment, a suitable gel is comprised of a semi-solid system inwhich a liquid phase is constrained within a three dimensional polymericmatrix with a high degree of cross-linking. The liquid phase may becomprised of water, together with the desired amount of UP256(bakuchiol), from 0 to 20% of water-miscible additives, for exampleglycerol, polyethylene glycol, or propylene glycol, and from 0.1 to 10%,preferably from 0.5 to 2%, of a thickening agent, which may be a naturalproduct, for example tragacanth, pectin, carrageen, agar and alginicacid, or a synthetic or semi-synthetic compound, for examplemethylcellulose and carboxypolymethylene (carbopol); together with oneor more preservatives, for example from 0.1 to 2% of methyl4-hydroxybenzoate (methyl paraben) or phenoxyethanol-differential.Another suitable base, is comprised of the desired amount of UP256(bakuchiol), together with from 70 to 90% of polyethylene glycol (forexample, polyethylene glycol ointment containing 40% of polyethyleneglycol 3350 and 60% of polyethylene glycol 400, prepared in accordancewith the U.S. National Formulary (USNF)), from 5 to 20% of water, from0.02 to 0.25% of an anti-oxidant (for example butylated hydroxytoluene),and from 0.005 to 0.1% of a chelating agent (for example ethylenediaminetetraacetic acid (EDTA)).

The term soft paraffin as used above encompasses the cream or ointmentbases white soft paraffin and yellow soft paraffin. The term lanolinencompasses native wool fat and purified wool fat. Derivatives oflanolin include in particular lanolins which have been chemicallymodified in order to alter their physical or chemical properties andsynthetic equivalents of lanolin include in particular synthetic orsemisynthetic compounds and mixtures which are known and used in thepharmaceutical and cosmetic arts as alternatives to lanolin and may, forexample, be referred to as lanolin substitutes.

One suitable synthetic equivalent of lanolin that may be used is thematerial available under the trademark Softisan™ as Softisan 649.Softisan 649, available from Dynamit Nobel Aktiengesellschaft, is aglycerine ester of natural vegetable fatty acids, of isostearic acid andof adipic acid; its properties are discussed by H. Hermsdorf in Fette,Seifen, Anstrichmittel, Issue No. 84, No. 3 (1982), pp. 3-6.

The other substances mentioned hereinabove as constituents of suitableointment or cream bases and their properties are discussed in standardreference works, for example pharmacopoeia. Cetomacrogol 1000 has theformula CH₃(CH₂)m(OCH₂CH₂)_(n)OH, wherein m may be 15 or 17 and n may be20 to 24. Butylated hydroxytoluene is 2,6-di-tert-butyl-p-cresol.Nipastat is a mixture of methyl, ethyl, propyl and butyl4-hydroxybenzoates.

The compositions of the invention may be produced by conventionalpharmaceutical techniques. Thus the aforementioned compositions, forexample, may conveniently be prepared by mixing together at an elevatedtemperature, preferably 60-70° C., the soft paraffin, liquid paraffin ifpresent, and lanolin or derivative or synthetic equivalent thereof. Themixture may then be cooled to room temperature, and, after addition ofthe hydrated crystalline calcium salt of mupirocin, together with thecorticosteroid and any other ingredients, stirred to ensure adequatedispersion.

Regardless of the manner of administration, the specific dose iscalculated according to the approximate body weight of the host. Furtherrefinement of the calculations necessary to determine the appropriatedosage for treatment involving each of the above mentioned formulationsis routinely made by those of ordinary skill in the art and is withinthe scope of tasks routinely performed by them without undueexperimentation, especially in light of the dosage information andassays disclosed herein. These dosages may be ascertained through use ofthe established assays for determining dosages utilized in conjunctionwith appropriate dose-response data.

It should be noted that the invention described herein may be used forveterinary as well as human applications and that the term “host” shouldnot be construed in a limiting manner. In the case of veterinaryapplications, the dosage ranges can be determined as described above,taking into account the body weight of the animal.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Method for the Quantification of Bakuchiol, Psoralen andIsopsoralen by HPLC

The amount of bakuchiol, psoralen and isopsoralen in the extracts,fractions, and the novel composition generated as described below wasquantified by a high pressure liquid chromatography (HPLC) using aPhotoDiode Array detector (HPLC/PDA) and a Luna Phenyl-hexyl column (250mm×4.6 mm). The targeted compounds were eluted from the column using anacetonitrile (ACN) water gradient from 36% to 100% ACN over a period of12 minutes, followed by 100% ACN for three minutes. The detailed HPLCconditions used are set forth in Table 1. A chromatogram of the HPLCseparation is depicted in FIG. 1. The targeted compounds were identifiedand quantified based on retention time and UV peak area usingcommercially available pure bakuchiol, psoralen and isopsoralen asquantification standards. The retention times for the bakuchiol,psoralen and isopsoralen were 18.19 minutes, 7.33 minutes and 7.95minutes, respectively.

TABLE 1 HPLC Conditions for quantification of Bakuchiol, Psoralen andIsopsoralen Column Luna Phenyl-hexyl, 150 × 4.6 mm Gradient 0-8 min 36%ACN/water 8-20 min 36% ACN/water to 100% ACN 20-23 min 100% ACN 23-28min 36% ACN/water Flow rate 1 mL/min Detection 0-11 min 246 nm (forpsoralen and angelicin, 7-8 min) 11-28 min 260 nm (for bakuchiol, 18-19min) Temperature 35° C. Standard concentration 0.1 mg/mL in MeOH forbakuchiol 0.025 mg/mL for psoralen and angelicin Extract preparation 0.2mg/mL in MeOH Linear range 0.01 mg/mL to 0.15 mg/mL

Example 2 General Methods for the Extraction of Bakuchiol from PsoraleaPlants Wrist Shaker

To a flask was added solvent (100 mL) and Psoralea corylifolia seedpowder (10 g) and the mixture was shaken on a wrist shaker at roomtemperature for one hour. The mixture was then passed through a filterand the filtrate collected. The extraction process was repeated one moretime with fresh solvent, the filtrates were combined, the solventremoved on a rotoevaporator and the residue was dried under high vacuum.

Reflux

To a flask was added solvent (50 mL) and Psoralea corylifolia seedpowder (10 g) and the mixture was refluxed for 40 min. The solution wasthen filtered and the extraction process was repeated two more timeswith fresh solvent. The filtrates were combined and the solvent wasevaporated to obtain a dried extract.

Following above extraction methods, sample plant material was extractedwith the following solvents: dichloromethane (DCM), EtOAc, acetone,MeOH, petroleum ether (BP 35-60° C.) and petroleum ether (BP 60-90° C.).The extracts were then analyzed by HPLC analysis as described inExample 1. The results are set forth in Table 2.

TABLE 2 Quantification of various Psoralea corylifolia extractsPetroleum Ether Petroleum Ether Petroleum Ether (35-60° C.) DCM EtOAcAcetone MeOH (35-60° C.) (60-90° C.) Extract wt. 0.5833 1.7535 1.67101.8932 1.8795 0.6457 0.9203 (g) % 29.1% 14.2% 13.7% 13.7% 13.9% 25.6%27.2% Bakuchiol in Extract % 1.7% 2.5% 2.3% 2.6% 2.6% 2.6% 2.7%Bakuchiol in Plant Method Wrist shaker (100 ml/10 g solid) Reflux (50ml/10 g solid)

Example 3 Large Scale Extraction of Bakuchiol from Psoralea Plants

Seed powder of Psoralea corylifolia (2 kg) and 9 liter of petroleum(pet.) ether (BP 60-90° C.) were rotated in a 20 L flask on arotoevaporator at 70° C. in a water bath for 1 hour. The solution wasthen decanted into a separate container and the solvent was removedunder vacuum. Fresh solvent was added into the biomass and theextraction process was repeated three more times. The extracts werecombined and evaporated to yield 335 g of a crude extract (MH-258-01-01)having 21% bakuchiol and 3% psoralen/isopsoralen by weight.

Example 4 Evaluation of Various Chromatographic Methods for PurifyingBakuchiol Extracts

Various chromatographic methods for purifying the crude solvent extract(MH-206-70-1) isolated from the seeds of Psoralea corylifolia using themethod described in Example 2, were evaluated to determine the potentialfor using column chromatography as a means of obtaining high puritybakuchiol free of contamination by furanocoumarins, particularlypsoralen/isopsoralen contamination. Briefly, each empty column cartridge(1.3 cm ID and 20 mL capacity, from Bio-Rad) was packed with a differentand eluted with different solvents in an attempt to separate thefuranocoumarin impurities from bakuchiol. The fractions (10 mL perfraction) were collected in test tubes and analyzed with silica gel TLCplates developing with 20% EtOAc/petroleum ether. The targetedcompounds, bakuchiol, psoralen and isopsoralen were identified based ontheir retention times, which were determined using standard solutions.The results are set forth in Table 3.

TABLE 3 Summary of column chromatographic separation of bakuchiol fromfuranocoumarins in crude extracts of Psoralea corylifolia Column size/Media Extract Loading Elution Solvent Results Al₂O₃ (neutral) 2 mL/25mg 1. petroleum ether Little separation (J. T. Baker) 2. EtOAc 3. MeOHXAD-4 (amerlite 5 mL/19 mg MeOH/water gradient in No separationpolystyrene 20% increments from 100% resin) water to 100% MeOH XAD-7(amerlite 8 mL/16 mg pet. ether/EtOAc gradient in Some separationpolyacrylate 20% increments from 100% resin) petroleum ether to 100%MeOH MeOH/water gradient in Little separation 20% increments from 100%water to 100% MeOH Polyamide 5 mL/50 mg 1. petroleum ether No separation2. 5% acetone/pet. ether 3. acetone LH-20 8 mL/50 mg petroleum ether Noseparation Silica gel 5 mL/50 mg 1. petroleum ether Good separation 2.15% EtOAc/pet. ether CG-71md 5 mL/50 mg 1. petroleum ether No separation2. acetone CG-161cd 5 mL/50 mg petroleum ether No separation 6 mL/50 mgMeOH/water step gradient Good separation Low yield

Example 5 Hydrolysis of a Petroleum Ether Extract Isolated from theSeeds of Psoralea corylifolia

A petroleum ether extract (25 g; MH-258-01-01), isolated from the seedsof Psoralea corylifolia as described in Example 3, was mixed with 500 mLof a NaOH solution (56.5 mM) in a 1 L round bottom flask. The solutionwas refluxed in a heating mantel for one hour. A small portion of thesolution was taken from the flask periodically and analyzed by HPLC asdescribed in Example 1. The reaction was stopped after HPLC analysisshowed that the peaks for psoralen and isopsoralen had completelydisappeared. The reaction mixture was then cooled to room temperature toyield a dark brown solution having a solid content of approximately 36mg/mL (MH-258-10-01).

To 20 mL of the hydrolysis solution (MH-258-10-01) in a separatoryfunnel (150 mL) was added DCM (20 mL). The mixture was shaken for 10 minand the layers were allowed to separate. The DCM layer was removed fromthe bottom of the separatory funnel and the extraction was repeated onemore time with 20 mL of fresh DCM. The organic layers were combined, andthe solvent removed by rotary evaporation under vacuum to yield 118 mgof a composition (MH-258-07-01), which contained 31% bakuchiol and wasfree of furanocoumarin contaminants (FIG. 3).

To 20 mL of the hydrolysis solution (MH-258-10-01) in a separatoryfunnel (150 mL) was added petroleum ether (20 mL). The mixture wasshaken for 10 min. and the layers were allowed to separate. Thepetroleum ether layer was removed from the top of the separatory funneland the extraction was repeated one more time with 20 mL of freshpetroleum ether. The organic layers were combined and the solventremoved by rotary evaporation under vacuum to yield 136 mg of acomposition (MH-258-07-02), which contained 41% bakuchiol and was freeof furanocoumarin contaminants (FIG. 4).

Example 6 Hydrolysis of a Methanol Extract Isolated from the Seeds ofPsoralea corylifolia

A dried methanol extract (MH-293-68-01), isolated from the seeds ofPsoralea corylifolia as described in Example 2, was mixed with 1 L of aNaOH solution (44 g NaOH in DI water) in a 2 L beaker on a stirrer/hotplate. The solution was stirred while boiling for 2 hours. Water wasadded to the beaker as necessary to maintain the total volume at about1200 mL. After 2 hours the solution was allowed to cool to roomtemperature, after which time 600 mL was transferred to a separatoryfunnel. Petroleum ether (250 mL) was added and the mixture was shakenfor 10 min. and the layers were allowed to separate. The petroleum etherlayer was removed from the top of the separatory funnel and theextraction was repeated (3×) with fresh solvent. The organic extractswere combined and the solvent removed by rotary evaporation under vacuumto yield 2.25 g of a composition (MH-293-74-01), which contained 51%bakuchiol and was free of furanocoumarin contaminants.

Example 7 Hydrolysis of Petroleum Ether Extract Isolated from the Seedsof Psoralea corylifolia Followed by Purification by ColumnChromatography

A petroleum ether extract (25 g; MH-258-01-01), isolated from the seedsof Psoralea corylifolia as described in Example 3, was mixed with 500 mLof a NaOH solution (56.5 mM) in a 1 L round bottom flask. The solutionwas refluxed for one hour. A small portion of the solution was takenfrom the flask periodically and analyzed by HPLC as described inExample 1. The reaction was allowed to proceed until HPLC analysisshowed that the peaks for psoralen and isopsoralen had completelydisappeared, The reaction mixture was then cooled to room temperature toyield a dark brown solution having a solid content of approximately 36mg/mL (MH-258-10-01).

150 mL of this solution (MH-258-10-01) was loaded onto a pre-preparedCG-161cd column. The pre-prepared column (5×13 cm) contained 300 mL ofCG-161cd resin, which had been equilibrated with 4 column volumes of DIwater. The loading material (MH-258-10-01) was fed into the top of thecolumn and eluted with 2500 mL of DI water to bring the column to pH 7,followed by 2500 mL of 70% MeOH/water and 4500 mL of 90% MeOH/water toelute bakuchiol. The eluent was monitored by TLC until the bakuchiol wascompletely eluted from the column. Fractions containing only bakuchiol(2 L) were combined and evaporated under vacuum to remove the solvent.Using this method highly pure bakuchiol (2.3 g) (99% pure, MH-258-12-08and -09), free of furanocoumarin contamination was obtained. (See FIG.5). Earlier and later bakuchiol fractions were also collected, combinedand evaporated under vacuum. From these fractions a composition(MH-258-12-07 and -10) was obtained in a quantity of 2.4 grams, whichcontained 70% bakuchiol free of furanocoumarin contamination. Theloading capacity of the CG-161cd column was estimated as 400 mL of thecrude hydrolysis solution per liter of CG-161cd resin. After separation,the CG-161cd column was recovered by washing with Clorox followed withMeOH and 4 column volumes of DI water. It can be reused for a number ofyears.

Example 8 Inhibition of COX-1 and COX-2 by Purified Bakuchiol

In order to screen for compounds that inhibited COX-1 and COX-2activity, a high throughput, in vitro assay was developed that utilizedthe inhibition of the peroxidase activity of both enzymes. (Needleman etal. (1986) Annu Rev Biochem. 55:69). Briefly, the composition orcompound being examined was titrated against a fixed amount of COX-1 andCOX-2 enzymes. A cleavable, peroxide chromophore was included in theassay to visualize the peroxidase activity of each enzyme in presence ofarachidonic acid as a cofactor. Typically, assays were performed in a96-well format. Each inhibitor, taken from a 10 mg/mL stock solution in100% DMSO, was tested in triplicate at room temperature using thefollowing range of concentrations: 0, 0.1, 1, 5, 10, 20, 50, 100, and500 μg/mL. To each well, 150 μL of 100 mM Tris-HCl, pH 7.5 was addedalong with 10 μL of 22 μM Hematin diluted in tris buffer, 10 μL ofinhibitor diluted in DMSO and 25 units of either the COX-1 or COX-2enzyme. The components were mixed for 10 seconds on a rotating platform,followed by the addition of 20 μL of 2 mMN,N,N′N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) and 20 μLof 1.1 mM arachidonic acid to initiate the reaction. The plate wasshaken for 10 seconds and then incubated 5 minutes before reading theabsorbance at 570 nm. The inhibitor concentration vs. % inhibition wasplotted and the IC₅₀ determined by taking the half-maximal point alongthe isotherm and intersecting the concentration on the X-axis. The IC₅₀was then normalized to the number of enzyme units in the assay. A highpurity (99%) bakuchiol sample (MH-258-08 was tested for both COX-1 andCOX-2 inhibition. The results are summarized in the following Table 4.

TABLE 4 Inhibition of COX activity by bakuchiol Compound Name COX-1(IC₅₀) COX-2 (IC₅₀) MH-258-08 2.34 μM 5.78 μM (99% bakuchiol)

Example 9 Inhibition of 5-Lipoxygenase by Purified Bakuchiol(MH-258-12-08)

As noted above, one of the most important pathways involved in theinflammatory response is produced by non-heme, iron-containinglipoxygenases (5-LOX, 12-LOX, and 15-LOX), which catalyze the oxidationof fatty acids such as AA to produce the hydroperoxides 5-, 12- and15-HPETE, which are then converted to leukotrienes. A LipoxygenaseInhibition Assay was carried out using a published method (Carter et al.(1991) J. Pharmacol. Exp. Ther. 256(3):929-937, Safayhi et al. (2000)Planta Medica. 66:110-113). 5-LOX was isolated from human PBML cells andarachidonic acid was utilized as a substrate. The test article andpositive control were dissolved in 1% DMSO. HBSS (Hank's balanced saltsolution) was used as incubation buffer, Pre-incubation time was 15minutes at 37° C., followed by 15 min. incubation at the sametemperature. This assay detects the formation of LTB4 with EIAquantification. Highly pure bakuchiol (99% bakuchiol, #MH-258-12-08) wastested in duplicate at concentrations of 10 μM, 1 μM, 0.1 μM, and 10 nMrelative to a positive control—NDGA at five concentrations. The doseresponse curve is illustrated in the FIG. 6. The IC₅₀ for 5-LOXinhibition by bakuchiol (99% pure) was 3.41 μM.

Example 10 Antimicrobial Activity of Purified Bakuchiol

The anti-microbial activity of a highly pure bakuchiol sample (99% pure;#MH-258-12-08) was evaluated using published methods (Modugno et al.(1994) Antimicrobial Agents & Chemotherapy 38:2362-2368; Misiek et al.(1973) Antimicrobial Agents & Chemotherapy 3:40-48). Briefly,Staphylococcus epidermidis (Gram Positive, ATCC 12228) was cultured for20 hours at 37° C. in Mueller-Hinton Broth medium. Propionibacteriumacnes (ATCC 6919) was cultured for 2 days at 37° C. in ReinforcedClostridial medium. The test article and positive control were dissolvedin 1% DMSO with an incubation volume of 1 mL. The time of assessment was1 day. Measurement of turbidity was used as the method ofquantification. A highly pure bakuchiol sample (99%; #MH-258-12-08) wastested in duplicate at concentrations of 100 μg/mL, 30 μg/mL, 10 μg/mL,3 μg/mL, 1 μg/mL, 0.3 μg/mL, 0.1 μg/mL and 0.03 μg/mL relative topositive controls—gentamicin at 0.1 μg/mL for Staphylococcus epidermidisand ampicillin at 0.1 μg/mL for Propionibacterium acnes, respectively.Significant inhibition was exhibited by bakuchiol at 1 μg/ml againstboth Staphylococcus epidermidis and Propionibacterium acnes. The highlypure sample of bakuchiol also inhibited the activity of Trichophytonmentagrophytes (ATCC 9533) at a moderate concentration of 30 μg/mL.Finally, no inhibition was observed for Epidermophyton floccosum (ATCC18397), Microsporum canis (ATCC 36299) and Pityrosporum ovale (ATCC38593).

Example 11 Evaluation of Acute Toxicity of Purified Bakuchiol

Acute Toxicity studies were completed testing two purity levels ofbakuchiol (UP256) (a sample that was approximately 20% pure and a samplewhich was 99% pure). Forty female ICR mice (Harlan) aged 4-5 weeks oldwere used for the 14-day study. Mice were administered 100 mL of theacute dose daily, approximately 2 g/kg by weight per test article perday. The first 10 mice received the composition containing 20.7%bakuchiol, while the second group of 10 mice received a compositioncontaining 99% bakuchiol. The UP256 composition was suspended in waterand administered through a syringe. Twenty mice were administered water,as the control group. The weights of all mice were measured, includingbaseline, 3 mid points, and an endpoint. Also, food and waterconsumption were observed for all groups. Any abnormal health conditionsor behavior was recorded over the two-week period. A necropsy of allmice was completed on day 14 and blood from all groups was collected fora complete blood screen. Two mice picked randomly from each of groupsalso had kidney and liver tissues removed for Histopathology. All bloodwork and pathology work was completed through Antech Diagnostics.

The average weight calculated for all groups, including controls,continued to increase over the two-week study period. No mouse showed adecrease in weight, and food/water consumption remained the same for allgroups. Aggression towards the investigator (i.e. biting) and towardseach other (i.e. fighting in cage) was the only significant differencein behavior among the treated mice compared to the control mice. This isa behavior that would be expected in the mice receiving androgen, a malehormone. The necropsy of all mice showed no gross abnormalities orchanges in any organ. The blood work showed the treated groups werenormal relative to the controls. The protein, enzyme, and ion levelswere within normal values for ICR mice. The kidney and liver tissueswere sent for Histopathology to assess any micro changes in the organsand the report stated that significant changes were not present in thekidney and the hepatocyte nuclei for the liver were within normal limitsfor mice. There was no substantial inflammation or evidence of neoplasiain either tissue section examined.

In conclusion, all weights, blood work, and histological data, was thesame for the treated mice relative to the control group. No adverseeffects were observed in the fourteen-day study for either sample ofUP256. Therefore, it can be concluded that UP256 at both purity levels(20% and 99% purity) has a solid safety profile

1-27. (canceled)
 28. A method for preparing a plant extract comprisingbakuchiol, the method comprising: a) extracting a plant or plant partcontaining bakuchiol with an organic solvent to obtain an organicextract comprising bakuchiol and furanocoumarin impurities; b) heatingthe organic extract obtained in step a) in the presence of a base at atemperature and for a time sufficient to hydrolyze a lactone ring of thefuranocoumarin impurities; and c) separating the hydrolyzedfuranocoumarin impurities from the bakuchiol to obtain the plant extractcomprising bakuchiol, wherein the plant extract is free offuranocoumarin impurities as measured by high pressure liquidchromatography (HPLC).
 29. The method of claim 28, wherein the plant orplant part is from the Psoralea genus of plants.
 30. The method of claim28, wherein the plant or plant part is from Otholobium pubescens(Fabaceae), Psoralea corylifolia L. (Luguminosae) or Psoralea glandulosaL. (Papilionaceae).
 31. The method of claim 28, wherein the plant partis seeds, stems, bark, twigs, tubers, roots, root bark, young shoots,rhixomes, flowers, leaves or combinations thereof.
 32. The method ofclaim 28, wherein the furanocoumarins are psoralen, isopsoralen orcombinations thereof.
 33. The method of claim 28, wherein step b)comprises refluxing.
 34. The method of claim 28, wherein the basecomprises a hydroxide base.
 35. The method of claim 34, wherein the baseis sodium hydroxide, potassium hydroxide, calcium hydroxide or lithiumhydroxide.
 36. The method of claim 28, wherein separating the hydrolyzedfuranocoumarin impurities from the bakuchiol comprises solventpartitioning.
 37. The method of claim 28, wherein the method furthercomprises steps of column chromatography, extraction followed bycrystallization, solvent partition, recrystallization or combinationsthereof.
 38. The method of claim 28, wherein the plant extract comprisesfrom 14 to 100 weight percent of bakuchiol.
 39. The method of claim 28,wherein the plant extract comprises from 30 to 100 weight percent ofbakuchiol.